Protein C is the precursor to activated protein C (APC), a potent natural anticoagulant. Protein C is activated by thrombin in complex with thrombomodulin (TM). The activation is augmented by endothelial cell protein C receptor (EPCR). TM and EPCR can be down-regulated due to inflammatory mediators, such as tumor necrosis factor, reviewed by Esmon (Esmon, C. T., “Natural anticoagulants and their pathways,” Handbook of Experimental Pharmacology 132:447–76; G. V. R. Born, Cuatrecasas, P., Ganten, D., Herken, H., Starke, K., and Taylor, P., eds., Springer-Verlag, New York 1999). TM and EPCR have also been found to be reduced in some forms of septic shock, meningococcemia in particular. Since EPCR and TM are expressed on endothelium, it is not possible to directly determine how well they are functioning without removal of blood vessels.
An alternative is to measure the levels of APC in circulation. Circulating APC levels can be measured with an enzyme capture assay. In this procedure, antibodies directed against protein C that cross react with APC are used to adsorb the protein C/APC from plasma. The antibodies can be coupled to affinity beads (Comp, P. C., R. R. Nixon, and C. T. Esmon. 1984. “Determination of protein C, an antithrombotic protein, using thrombin-thrombomodulin complex,” Blood 63:15–21), or placed on a plastic microtiter plate (Gruber, A. and J. H. Griffin. 1992., “Direct detection of activated protein C in blood from human subjects,” Blood 79:2340–48). The APC levels are also proportional to the protein C concentration in the plasma and to the thrombin concentration. The thrombin generation can be assessed either by measuring prothrombin fragment 1–2 levels or thrombin-antithrombin complex levels using ELISA based assays or radio-immuno assays. Protein C levels can be determined either by ELISA or a variety of functional assays that are commercially available.
Knowledge of the circulating levels of APC is important in evaluating the status of critically ill patients. Recently, APC has been shown to be effective in the treatment of septic shock. Previous studies have shown a correlation between decreases in protein C levels in septic patients and a negative clinical outcome (e.g., death, amputation, and organ failure). There are many clinical reports that supplementation with protein C can improve clinical outcome in patients with sepsis, particularly meningococcemia. (Yan, S. B. and J. F. Dhainaut. 2001. “Activated protein C versus protein C in severe sepsis,” Crit Care Med 29:S69–74; Mesters, R. M., J. Helterbrand, B. G. Utterback, B. Yan, Y. B. Chao, J. A. Fernandez, J. H. Griffin and D. L. Hartman. 2000. “Prognostic value of protein C concentrations in neutropenic patients at high risk of severe septic complications,” Crit Care Med 28:2209–2216; White, B., W. Livingstone, C. Murphy, A. Hodgson, M. Rafferty and O. P. Smith. 2000. “An open-label study of the role of adjuvant hemostatic support with protein C replacement therapy in purpura fulminans-associated meningococcemia,” Blood 96:3719–372400. We have found that in some meningococcemia patients, supplementation with protein C failed to elicit detectable APC generation, indicating that the protein C activation mechanism was compromised. This is consistent with the observation that the EPCR and TM levels were reduced on the vasculature (Faust, S. N., R. S. Heyderman, O. Harrison, R. D. Goldin, Z. Laszik, C. T. Esmon, and M. Levin, “Molecular mechanisms of thrombosis in meningococcal septicaemia: the role of the protein C pathway in vivo,” 2nd Annual Meeting of the British Infection Society, London, Apr. 23, 1999 (Abstr.)).
It would be very useful to be able to measure circulating APC, especially in clinical situations where a decision to use protein C versus APC in therapy needs to be expeditiously made, within a matter of hours at most. The currently available enzyme capture assay for APC using the microtiter plates is not useful for this purpose since the assay may take up to three weeks to develop. The major reasons for the very long times required in the enzyme capture assay are the low levels of circulating APC (about 3 ng/ml plasma) in normal individuals and the very high concentration of protein C (about 3000 ng/ml plasma) relative to the enzyme (Gruber, A. and J. H. Griffin. 1992., “Direct detection of activated protein C in blood from human subjects,” Blood 79:2340–48). With the relatively low capacity of the microtiter plates used in the assay, the plasma must be diluted approximately 30 fold or more to allow capture of most of the protein C and APC. This problem could be circumvented if the capture antibody exhibited a high degree of specificity toward APC.
We have now developed an antibody (hereinafter referred to as “HAPC 1555”) having a high degree of specificity toward APC. The antibody can detect APC in plasma within one hour, thus making the direct detection of APC practical clinically. Such antibodies can be adsorbed to surfaces including but not limited to microtiter plates. The antibody of the present invention exhibits improved capacity for APC, thus providing an improved assay for APC.